Expression vectors

ABSTRACT

Expression vectors encoding bacteriophage signal peptides are described. The vectors may be used for the heterologous expression and secretion of polypeptides such as antibodies in bacterial host cells.

[0001] The present invention relates to nucleotide sequences encodingbacteriophage signal peptides, expression vectors containing suchsequences and their use for the heterologous expression and secretion ofpolypeptides, in particular antibodies, in bacterial systems.

[0002] Throughout this application various publications are referencedby author and year. Full citations for these publications are providedfollowing the detailed description of the invention and Examples.

[0003] Recombinant protein production has been facilitated to a largeextent by the construction of expression systems that are capable ofexporting the protein of interest from the cell in which it isexpressed. In order to effect secretion of the recombinant product fromthe host cell, these expression systems utilise amino-terminal peptideextensions, or signal peptides, which are found on the majority ofeukaryotic and prokaryotic proteins that are destined for export fromthe cytoplasm. The characterisation of a number of signal peptides fromdiverse sources has revealed that whilst there is little sequencehomology amongst them, they do share certain functional characteristics.These common features are a positively charged amino-terminal region, acentral hydrophobic core, and a more polar carboxy-terminal region thatnormally terminates with a signal peptidase cleavage site.

[0004] It is quite common that the signal peptides employed in suchexpression systems are native to the expression host, for example, thePhoA, MalB and OmpA signal peptides of Escherichia coli have been usedextensively to secrete polypeptides to the periplasm of that organism.However, some signal peptides are capable of working even when movedbetween species, e.g. secretion of Human growth hormone to the E. coliperisplasm was more efficient when the native signal peptide wasemployed (Gray et al, 1985), and rice α-amylase has been efficientlysecreted from Saccharomyces cerevisiae using its native signal sequence(Kumagai et al., 1990).

[0005] Unfortunately, the efficacy of individual signal peptides indifferent systems is unpredictable. The process is frequentlyinefficient, with low yields being commonplace. In addition, problemsmay be encountered with the misprocessing of the signal peptide, whichmay be improperly removed or incompletely cleaved. Thus, there is a needfor signal peptides, which can direct secretion consistently in anefficient and universal manner i.e. achieve high yields and/or accuratecleavage.

[0006] We have found, unexpectedly, that we can obtain high levels ofsoluble polypeptides, in particular antibodies or antigen bindingfragments thereof, from prokaryotic cells when we use a bacteriophagesignal sequence (for example, that of the bacteriophage M13 major coatprotein) to mediate the secretory process.

[0007] Thus according to the first aspect of the invention we provide amethod of producing an antibody chain, or an antigen binding fragmentthereof, comprising culturing host cells containing an expressioncassette, under conditions that result in expression of the antibodychain, or antigen binding fragment thereof, from the expressioncassette, wherein the expression cassette comprises a first nucleic acidencoding a bacteriophage signal peptide, or a variant thereof, operablylinked to and in frame with a second nucleic acid encoding the antibodychain or antigen binding fragment thereof.

[0008] Antibodies are assembled from two light and two heavy chainpolypeptides, which are linked to each other through di-sulphide bonds.Thus the term “antibody chain” as used herein refers to either anantibody light chain polypeptide or an antibody heavy chain polypeptide.

[0009] The term “antigen binding fragment” as applied to an antibodychain, is herein defined as any fragment or domain of an antibody chainthat is capable of binding to an antigen independently and selectively.Examples of such antigen-binding fragments, which may be expressed andsecreted according to the method of the invention, include for example,V_(H) and V_(L) fragments, and single chain antibodies such as, forexample a scFv.

[0010] According to another aspect, the method of the invention may beused to produce a whole antibody comprising full-length heavy and lightchains, or fragments thereof including, for example, Fab, Fab′, F(ab′)₂,and Fv fragments This may be achieved by producing antibody light andheavy chains or appropriate fragments thereof, according to the methodof the invention, in different host cells, and then allowing assembly ofthe appropriate chains or fragments thereof to form a whole antibody orantibody fragment after the chains have been expressed.

[0011] Alternatively a whole antibody, or fragment thereof, may beproduced by introducing at least two expression cassettes into the samehost cell. Each expression cassette will comprise a first nucleic acidencoding a bacteriophage signal peptide (or a variant thereof). Thiswill be operably linked to and in frame with a second nucleic acid,which will encode an antibody heavy chain or appropriate heavy chainfragment in one expression cassette and an antibody light chain orappropriate light chain fragment in another expression cassette. Thus,heavy and light chains, or fragments thereof, may be co-expressed withinthe same cell and secretion of each may be mediated by a bacteriophagesignal peptide. Such expression cassettes may be introduced into hostcells as distinct entities that are incorporated within a single nucleicacid molecule, or alternatively they may be introduced on separatenucleic acid molecules.

[0012] Whole antibodies, which may be produced as described above,include multimeric monospecific antibodies, as well as bi-specific ormulti-specific antibodies.

[0013] An antibody or antigen binding fragment thereof, expressed andsecreted according to any aspect of the invention may be polyclonal or,especially monoclonal. It may belong to any immunoglobulin class and mayfor example be an IgG (for example IgG1, IgG2, IgG3 or IgG4), IgE, IgMor IgA antibody. It may be of animal, for example mammalian origin, forexample it may be a murine rat or human antibody or an antigen bindingfragment derived therefrom. Aftematively, the antibody or antigenbinding fragment may be chimeric i.e. contain portions derived fromdifferent animal species. Particular examples are well documented in theliterature and include CDR grafted antibodies and antigen bindingfragments.

[0014] Any signal peptide of bacteriophage origin may be employed in theinvention, however, it is preferred that the first nucleic acid encodesthe signal peptide from the bacteriophage M13 major coat protein, or avariant thereof. The term “variant” as used herein, refers to signalpeptides having substantially the same amino acid sequence as thatdescribed below for M13 major coat protein signal peptide and which arecapable of functioning at least as efficiently as the native M13 signalpeptide. This encompasses M13 major coat protein signal peptidederivatives that may have been modified to alter or enhance particularfeatures such as, for example, the signal peptidase recognition site. Asignal peptide that has “substantially the same amino acid sequence” isone that shares greater than 70% identity with the amino acid sequenceof the M13 major coat protein signal. Preferable variants will sharegreater than 75% identity, more preferably greater than 80% identity andmost preferably greater than 90% identity with signal peptide from theM13 major coat protein.

[0015] In order to assess the efficacy of the variant M13 major coatsignal peptides, they may be used to direct the secretion of a standardpolypeptide for example, β-lactamase or alkaline phosphatase. Any of thefollowing parameters—yield, rate of accumulation, accuracy ofcleavage—may then be measured and compared to those achieved by thenative M13 major coat protein signal peptide when used to directsecretion of the same model polypeptide. This provides the basis of asuitable screen for signal peptides that are capable of functioning atleast as efficiently, if not better than, the native M13 major coatprotein signal peptide. Methods of estimating yield and/or rate ofaccumulation will be obvious to the skilled man and may rely on directmeasurement of the polypeptide product, or alternatively they may relyon any intrinsic enzymatic activity of the polypeptide. Specificexamples of such methodology are described herein in more detail, in thedetailed description of the preferred embodiments.

[0016] The native bacteriophage M13 major coat protein signal peptide is23 amino acids in length and has the amino acid sequence“MKKSLVLKASVAVATLVPMLSFA”. Due to the degeneracy of the genetic code,any one of a number of nucleotide sequences may encode a signal peptidewith this sequence. Any of these nucleic acids including the nativebacteriophage M13 sequence, may be employed in the invention. In fact,we have shown, by altering the nucleotide sequence but not the aminoacid sequence, of the bacteriophage M13 major coat protein signalpeptide, the expression and secretion of soluble proteins in E. coli canbe optimised. Examples of such soluble proteins include enzymes (such asalkaline phosphatase); protein hormones or toxins; soluble transport,structural or contractile proteins and, in particular, antibodies.

[0017] Thus nucleic acids encoding the M13 major coat protein signalpeptide, which differ in the nucleotide sequence from the wild-type M13bacteriophage nucleic acid sequence, but do not differ in the amino acidsequence that they encode, form yet a further aspect of the inventionand may also be employed in methods of the invention as describedherein. The M13 major coat protein signal peptide, encoded by a nucleicacid according to this aspect of the invention may be employed asdesired, to direct the secretion of a full-length soluble protein, or afragment or domain thereof.

[0018] Nucleic acids according to this aspect of the invention maydiffer from the wild-type nucleotide sequence encoding the M13 majorcoat protein signal peptide in any number of nucleotide positionsprovided that the amino acid sequence that is encoded is not altered.Thus, a nucleic acid according to this aspect of the invention may onlydiffer in sequence at a single position, or alternatively it may differin sequence (from the wild type) in up to a maximum of approximately 31positions. Preferably such nucleic acids will differ in nucleotidesequence from the wild type in between approximately 18 to 25 positions.More preferably nucleic acids according to this aspect of the inventionwill differ in sequence from the wild type at a total of 20, 21, 22, or23 nucleotide positions.

[0019] Examples of preferred nucleic acid sequences encoding the M13major coat protein signal peptide for use in various aspects of theinvention include the native M13 nucleotide sequence (MCPn) and novelnucleotide sequences MCP1 to MCP9 given in Table 1 below. The use of anucleotide sequence corresponding to any one of MCPn, MCP1, MCP3, MCP4,or MCP8 is particularly preferred. TABLE 1 Nucleic acid sequencesencoding the signal peptide of the M13 major coat protein. Nucleotidesequence MCPn 5′ ATGAAAAAGTCTTTAGTCCTCAAAGCCTCTGTAGCCGTTGCTACCCTCGTTCCGATGCTGTCTTTCGCT 3′ MCP15′ ATGAAAAAAAGCCTGGTTCTGAAAGCGAGCGTGGCGGTGGCGACCCTGGTGCCGATGCTGAGCTTCGCG 3′ MCP2 5′ATGAAGAAAAGTCTTGTCCTGAAGGCGAGCGTGGCTGTAGCG ACGCTGGTGCCTATGCTGAGTTTCGCA3′ MCP3 5′ ATGAAGAAGAGTCTTGTGCTGAAGGCAAGTGTGGCAGTGGCTACGCTGGTGCCTATGCTGAGTTTTGCG 3′ MCP45′ ATGAAAAAAAGTCTTGTTCTGAAAGCAAGCGTGGCTGTAGCAACTCTTGTCCCTATGCTGAGTTTTGCG 3′ MCP55′ ATGAAGAAAAGTCTTGTACTGAAAGCGAGTGTGGCGGTCGCAACGCTGGTACCAATGCTGAGCTTCGCA 3′ MCP65′ ATGAAGAAGAGTCTTGTGCTCAAGGCAAGCGTAGCGGTGGCGACCCTCGTGCCGATGCTGAGTTTCGCG 3′ MCP75′ ATGAAGAAAAGTCTGGTACTGAAGGCGAGTGTGGCGGTGGCCACTCTGGTTCCAATGCTTAGTTTCGCG 3′ MCP85′ ATGAAGAAGAGTCTGGTGCTGAAAGCGAGTGTAGCGGTGGCAACGCTGGTGCCGATGCTGAGTTTTGCG 3′ MCP95′ ATGAAAAAGAGCCTGGTACTTAAGGCGAGTGTTGCGGTGGCGACGCTGGTCCCGATGCTGAGTTTTGCG 3′

[0020] Where it is desired that at least two polypeptides are to beproduced and secreted from a cell, it is preferable that at least twosignal peptide coding sequences described in Table 1 above, areemployed: one for each polypeptide. The signal peptide coding sequencemay be the same, or may be different for each polypeptide to besecreted. According to yet a further aspect of the invention there areprovided libraries containing random combinations of signal peptidecoding sequences. Example 5 describes such libraries in more detail.

[0021] By the term “operably linked”, it is meant that the nucleic acidsencoding both the signal peptide and the polypeptide that is to besecreted, are under the control of a single promoter/operator region andare transcribed as a single message. Thus, an expression cassette foruse in the invention can be, in its simplest form, the smallest geneticunit capable of mediating the expression and secretion of a polypeptideof interest. An expression cassette generally contains a suitablepromoter/operator region (including, for example, the tac or lac or T7or bacteriophage lambda promoter/operators for use in E. coli, theecdysone responsive or human cytomegalovirus or SV40 promoters for usein mammalian cells, the Gal1 or Cup1 or AOX1 promoters for use in yeastcells, and the polyhedrin promoter for use in baculovirus), upstream ofa 5′ untranslated region (5′UTR), which is in turn followed by a nucleicacid encoding the signal peptide and the polypeptide to be secreted.Expression cassettes may additionally incorporate the appropriatetranscriptional and translation control sequences, for example, enhancerelements, termination stop sequence, mRNA stability sequences, start andstop codons or ribosome binding sites, linked in frame with or includedwithin, where appropriate, the nucleic acid molecules of the invention.It may be desirable for the expression cassette to remain in an episomalform within the cell. Alternatively, it may integrate into the genome ofthe host cell. If the latter is desired, sequences that promoterecombination with the genome will be included in the expressioncassette. Accordingly, further aspects of the invention provide hostcells containing nucleic acids or expression cassettes as describedherein and/or expressing polypeptides according to the methods describedherein.

[0022] Nucleic acid sequences encoding the M13 major coat protein signalpeptide, or variants thereof, may find utility in any host. For example,such bacteriophage derived signal sequences may be of value in otherviral based expression systems such as, for example, baculovirus.However, prokaryotes, such as bacteria of the Streptomyces and Bacillusspecies, and E. coli are the preferred expression hosts. E. coli is aparticularly preferred host. Where the expression host is prokaryotic,the promoter/operator region of an expression cassette will be one thatis capable of regulating expression in prokaryotic hosts. As will beobvious to a person skilled in the art, if the signal peptides are to beemployed in other (e.g. eukaryotic) expression systems, thepromoter/operator regions will be capable of regulating expression inthe specific host.

[0023] The method of the invention may additionally comprise recoveringthe secreted polypeptide. If the expression host is a Gram negativebacterium, the secretion process may only take the polypeptide as far asthe periplasmic space. Where this is the case, the first step of anyrecovery procedure will be to harvest the cells (e.g. by centrifugation)and release the polypeptide from the periplasmic space. This may beachieved by disrupting the outer membrane, for example by osmotic shockor any by other suitable physically disruptive means, or by making useof host strains that have been genetically compromised and have a“leaky” outer membrane (e.g. certain strains of E. coli K12, see Atlan &Portarlier, 1984; Fognini Lefebvre & Portarlier, 1984). In otherexpression hosts, which lack an outer membrane, the polypeptide productmay be secreted directly into the culture medium.

[0024] Polypeptides that have been released from the periplasmic space,or secreted to the culture medium, may be recovered and purified furtherusing any suitable method. This includes any method which uses, forexample, a difference in solubility e.g. salting out and precipitationwith a solvent or, a difference in molecular weight e.g. ultrafiltrationand gel electrophoresis or, a difference in electric charge e.g. ionexchange chromatography or, specific affinity e.g. affinitychromatography or, a difference in hydrophobicity e.g. reverse phasehigh performance liquid chromatography or, a difference in isoelectricpoint e.g. isoelectric focusing, to aid purification’. Further detailsof suitable isolation procedures and protein purification strategieswill be familiar to the skilled artisan and are well documented in theart.

[0025] Nucleic acids for use herein may be generated using any standardmolecular biology and/or chemistry procedure, as will be clear to thoseof skill in the art. Particularly suitable techniques include theoligonucleotide directed mutagenesis of the native nucleic acid encodingthe M13 major coat protein signal peptide, oligonucleotide directedsynthesis techniques, and enzymatic cleavage or enzymatic filling in ofgapped oligonucleotides. Such techniques are described by Sambrook &Fritsch, 1989, and in the Examples contained hereinafter.

[0026] In further aspects, the nucleic acids or expression cassettes ofthe invention may be used with a carrier. The carrier may be a vector orother carrier suitable for the introduction of the nucleicacid/expression cassette into a host cell. Nucleic acids/expressioncassettes may be sub cloned into any suitable commercially availablevector (e.g. the pUC or pBluescript series of vectors for use in E.coli), using standard molecular biology techniques. Such vectors mayinclude plasmids, phagemids and viruses (including both bacteriophageand eukaryotic viruses). The invention includes both cloning andexpression vectors containing nucleic acids and/or expression cassettesof the invention. Where appropriate, such a vector or carrier maycontain more than one expression cassette according to the invention,for example, a Fab′ expression vector may contain one expressioncassette encoding an antibody light chain and one expression cassetteencoding an antibody heavy chain (see for example FIG. 1C).

[0027] Introduction of the nucleic acid or expression cassette into ahost cell may employ any available technique. In bacterial cells,suitable techniques may include calcium chloride transformation,electroporation or transfection using bacteriophage. In eukaryotic cellssuitable techniques may include calcium phosphate transfection, DEAEDextran, electroporation, particle bombardment, liposome mediatedtransfection or transduction using retrovirus, adenovirus or otherviruses, such as vaccinia or, for insect cells; baculovirus.

[0028] Following introduction of the nucleic acid into host cells, thecells may be cultured on a per se known medium suited for growing thehost, (for example 2xYT or LB Broth for E. coli). Any suitable mediumwill usually contain at least an assimable carbohydrate, a nitrogensource and essential minerals. The carbohydrates are usually in the formof simple sugars such as lactose or glucose, the nitrogen source mayinclude yeast extract or other sources of assimable amino acids such astryptone, casein, phytone, peptone and beef extract. The essentialminerals may vary between expression hosts but generally include traceamounts of transition metal salts such as manganese and magnesium salts.The culture medium may be modified, for example, by the addition of anantibiotic or other chemical or, by the exclusion of a particularnutrient, in order to maintain the presence of a vector or carrierwithin the host organism.

[0029] Growth conditions (e.g. growth medium, temperature, time andlength of induction and quantity of inducing chemical if the promoter isinducible) will vary according to the individual expression systememployed, but in general will be optimised in order enhance expressionof the recombinant polypeptide. For example, they may be manipulated inorder to allow accumulation of the expressed polypeptide in, forexample, the periplasmic space or culture medium. Allowing accumulationof the polypeptide product following expression, may form an optionalstep in the method of the invention. General guidance with respect togrowth and induction conditions, suitable for recombinant polypeptideexpression, may be found in the art (see for example, Sambrook &Fritsch, 1989; Glover, 1995a,b) and specific examples of cell cultureconditions and induction regimes are described herein in the followingExamples.

[0030] The various aspects and embodiments of the present invention willnow be illustrated in more detail by way of example. It will beappreciated that modification of detail may be made without departingfrom the scope of the invention.

BRIEF DESCRIPTION OF THE FIGURES

[0031]FIG. 1: Schematic Representation of Alkaline Phosphatase, scFv andFab′ Expression Cassettes. In part A) the arrangement of the alkalinephosphatase expression cassette is shown, with the signal peptide codingregion (SP) ligated in-frame with the alkaline phosphatase structuralgene. These are under control of the tac promoter (pTac). The positionof the 5′ untranslated region (5′ UTR) is also shown. In part B) thearrangement of the scFv expression cassette is shown. The signal peptide(SP) is ligated in front of and in-frame with the V_(L) coding sequence,which is subsequently linked in-frame with the V_(H) coding sequence viaa (Gly₄Ser)₄ linker (not shown). Expression is controlled via the tacpromoter (pTac) and the position of the 5′ untranslated region is alsoshown. In part C) the arrangement of the expression cassettes in theFab′ 40.4 expression construct is shown. Both the V_(L) and V_(H) codingsequences are each fused in-frame with the same signal peptide (SP)coding sequence. These are under the control of separate tac promoters(pTac). The position of the two 5′ untranslated regions (5′UTR) areshown, as is the cKappa intergenic spacer (C_(κ)), which separates lightand heavy chain expression. The C_(H1) coding region is shown fusedin-frame and downstream of the V_(H) coding region.

[0032]FIG. 2 Constructs to enable the production of Fab′-signal peptidelibraries. A] The second copy of the tac promoter was removed as an XhoI-Xba I fragment and replaced with a Xho I-Xba I fragment carrying a tacpromoter that had been modified to remove the internal Pst I and includeMfe I and Nsi I sites. B] shows the modified construct.

[0033]FIG. 3 Yield of Fab′ from constructs with different signal peptidecoding sequences in font of Heavy and Light chains The yield of Fab′ 165from various members of the signal peptide library having a heavy chainexpression cassette followed by a light chain expression cassette, wasassessed by ELISA two hours after the induction of expression. Resultsshown are the mean of three small scale shake flask experiments, ±SD.

[0034]FIG. 4 Effect of different signal peptide coding regions on heavychain, light chain and total Fab′ yield in fermentation. Yield wasassessed by surface plasmon resonance for samples taken at 2, 13, 20 and38 hours after the induction of expression, and is represented in termsof resonance units (RU). Data is shown for various members of the twosignal peptide libraries i.e. members of the library containing a lightchain expression cassette followed by a heavy chain expression cassette(LC:HC) and members of the library containing a heavy chain expressioncassette followed by a light chain expression cassette (HC:LC). SPCDS=signal peptide coding sequence.

EXAMPLES Example 1 Cloning of the Alkaline Phosphatase Gene

[0035] The phoA gene was cloned from E. coli strain W3110 by PCR, withits own signal peptide using primers PhoA1 (5′GCGCGCGCTCTGCAGGTCGAGTTCTAGATAACGAGGCGTAAAAAATGAAACAAAGCACTATTGCACTGGC3′) and PhoA2 (5′GCGCGCGCGCGGCCGCTCATTATTTCAGCCCCAGAGCGGCTTTCATGG 3′).The initiation codon of the native alkaline phosphatase gene was changedfrom GTG to the more common initiation codon of ATG. The PhoA1 primerincorporates a Pst I restriction endonuclease site at its 5′ end,followed by a “5′ UTR” region, the altered initiation codon and afurther 23 bases of the phoA gene. The PhoA2 primer incorporates a Not Irestriction endonuclease site at the 3′end of the phoA PCR product. Thispermits cloning of the Pst I-Not I double-digested PCR product behindany suitable promoter to form an expression cassette with the nativealkaline phosphatase signal peptide.

[0036] The mature phoA gene (without its signal peptide) was cloned fromE. coli strain W3110 using primers PhoA3 (5′CGGACACCAGAAATGCCTGTTCTGGAAAAC 3′) and PhoA2 (as above). This allows thecloning of the mature alkaline phosphatase gene behind any of the signalpeptide cassettes described herein, as a blunt-ended/Not I fragment.Thus the efficacy of variant signal peptides and/or different nucleicacids encoding the same signal peptide amino acid sequence may becompared using alkaline phosphatase as a standard protein.

Example 2 Construction of M13 Major Coat Protein Signal PeptideCassettes

[0037] Nucleic acid cassettes encoding the M13 major coat protein signalpeptides identified in Table 1 were constructed from pairs of longcomplementary oligonucleotides, which were annealed at a concentrationof 1 pmole/μl in buffer (25 mM NaCl, 12.5 mM Tris-HCl, 2.5 mM MgCl₂,0.25 mM DTE, pH 7.5) by heating in a boiling water bath for 5 minutesand then allowing them to cool slowly to room temperature.

[0038] The design of these oligos was such that they consist of threeelements: an upstream 5′ UTR region, a core encoding the signal peptideand a downstream linker region to permit subsequent cloning of thesignal peptide into an expression cassette in front of the gene encodingthe polypeptide that was to be secreted. As the skilled man willappreciate, the sequence of this linker may be varied in order to adaptthese signal peptide cassettes for use with other polypeptides.Cassettes encoding signal peptides that were used to direct secretion ofalkaline phosphatase lacked the downstream linker and consisted of the5′ UTR and the core signal peptide coding region only.

Example 3 Construction of Expression Vectors with the M13 Major CoatProtein Signal Peptide Encoded by Different Nucleic Acids

[0039] Alkaline phosphatase expression vectors: Signal peptide cassettesthat had been constructed by annealing oligonucleotides (see Example 2above), were ligated to Not I digested mature PhoA PCR product(described in Example 1 above), producing a Pst I-Not I fragment. Thiswas then ligated into vector behind the tac promoter (see FIG. 1A).

[0040] scFv expression vectors: To enable the facile introduction of newsignal peptide coding regions, an EcoR V restriction endonuclease sitewas introduced in the first two codons of the V_(L) domain of anexisting scFv expression plasmid [see for example the InternationalPatent Specification No. WO 01/94585] This plasmid contains a scFvspecific for a human cytokine, in the V_(L)-V_(H)-His organisation,under control of the tac promoter. The scFv also contains a (Gly₄Ser)₄linker. Signal peptide cassettes that had been constructed by annealingoligonucleotides (see Example 2 above) were then ligated into Pst I-EcoRV double digested scFv expression vector (see FIG. 1B).

[0041] Fab′ expression vectors: Vectors for expressing Fab′ light andheavy chains under the dual control of tac promoters were constructed asfollows. The V_(L) expression cassette within an existing Fab′expression vector [see for example the International PatentSpecification No. WO 01/94585] was excised by double digestion with PstI and Spl I restriction endonucleases, and replaced by SP-V_(L)cassettes, that had been similarly excised from the scFv expressionvectors described above. The SP-V_(H) fragments were created andintroduced as PCR fragments using a short reverse 3′ oligonucleotidethat anneals in the C_(H1) region, and long 5′ forward oligonucleotidesthat encode the signal peptide. This resulted in a series of Fab′ 40.4expression vectors containing the different signal peptides in front ofthe 40.4 light and heavy chains. These vectors were advantageouslydesigned such that there are unique restriction sites at the 5′ and 3′borders of the V_(L) and V_(H) regions (EcoR V-Spl I, Pvu II-Apa I)respectively, thus enabling rapid exchange of variable domain and/orsignal peptide coding regions (see FIG. 1C).

[0042] Control expression vectors for both Fab′ and scFv wereconstructed by replacing the M13 signal peptides with the E. coli OmpAsignal peptide.

Example 4 Heterologous Polypeptide Expression and Secretion Using M13Major Coat Protein Signal Peptide

[0043] a) Methods

[0044] Polypeptide Production in Liquid Culture—Production of AlkalinePhosphatase and scFv in Shake Flask.

[0045] Shake flask experiments and extraction of periplasmic fractionswere conducted essentially as described previously (Humphreys et al1996), with tetracycline being employed at a final concentration of 10μg/ml in the growth medium. Polypeptide expression was induced by theaddition of IPTG to 0.2 mM and assayed, either by enzyme assay or ELISAas appropriate, at time points between 0 and 5 hours post-induction.

[0046] Polypeptide Expression Liquid Culture—Production of Fab′ byFermentation.

[0047] Fermentations were run in media ‘SM6E’: (NH₄)₂SO₄ 5.2 gL⁻¹;NaH₂PO₄.H₂O 4.14 gL⁻¹; KCl 4.025 gL⁻¹; MgSO₄.7H₂O 1.04 gL⁻¹; citric acid5.20 gL⁻¹; glycerol 31.111 gL⁻¹; CaCl₂.2H₂O 0.0522 gL⁻¹; ZnSO₄.7H₂O0.0206 gL⁻¹; MnSO₄.4H₂O 0.0272 gL⁻¹; CuSO₄.5H₂O 0.0081 gL⁻¹; CoSO₄.7H₂O0.0042 gL⁻¹; FeCl₃.6H₂O 0.1006 gL⁻¹; H₃BO₃ 0.0003 gL⁻¹; Na₂MoO₄.2H₂O0.0002 gL⁻¹; MAZU DF843 as an antifoam at 0.02% (v/v), and the pH madeto 6.95 with NH₄OH. Fermentors (Braun BiostatB 2.5L) were inoculatedwith sufficient seed culture (in SM6E media supplemented withtetracycline at 10 μgml⁻¹) to give an initial OD₆₀₀ of 0.2. The pH wascontrolled by the addition of 50% (v/v) NH₄OH and 1.8M H₂SO₄ asnecessary, and the dissolved oxygen was maintained at 30% using variableagitation and airflow. Cultures were batch fed with 2×45 ml 80% (w/w)glycerol at OD₆₀₀ 20 and 40 respectively. Fab′ expression was induced atan OD₆₀₀ of approximately 80 by exhaustion of glycerol and substitutionof lactose as the carbon source. Lactose concentration was maintained atbetween 20 and 50 gL⁻¹ throughout the production phase, and cells wereharvested 24-36 hours post-induction.

[0048] Fermentation cell pastes were resuspended in ½ harvest volume of100 mM Tris.HCl/10 mM EDTA pH7.4 and agitated at 250 rpm, 30° C. for 16hours. Periplasmic extracts were clarified by centrifugation at 25,000 gfor 30 minutes and passed through a 0.2 μm filter (Millipore), beforepurification on Protein G Sepharose (GammaBind Plus, Pharmacia Biotech)as described previously (Humphreys et al., 1998).

[0049] Assay for Alkaline Phosphatase Activity

[0050] Assays were performed as essentially as described previously(Humphreys et al., 1995), with the following modifications: expressionwas induced by 0.2 mM IPTG, and assays were performed on 20 μl ofculture approximately 3 hours post induction. Alkaline phosphataseactivity was expressed as ΔA₄₂₀ OD₆₀₀ ⁻¹ min⁻¹.

[0051] ELISA of scFv and Fab′ Concentration in Shake Flask andFermentation Periplasmic Extracts.

[0052] For scFv ELISA Nunc Maxisorp plates were coated with antigen (ahuman cytokine) at 0.5 μgml⁻¹ in 100 mM sodium bicarbonate buffer pH 9.0for 16 hours at 4° C. After washing 4 times in blocking buffer (0.1% w/vBSA in PBS), and twice in glazing buffer (10% w/v trehalose, 0.1% w/vBSA in PBS) the plates were air dried and stored in sealed foil pouchesat 4° C. Purified standard was diluted to 250 ngml⁻¹, followed by serialtwo-fold dilutions, in 1% w/v BSA in PBS. Each well was incubated with100 μl of sample or standard and agitated at room temperature for 1hour. After washing twice with 0.0002% w/v Tween20 in PBS, each well wasincubated with 100 μl of rabbit anti-His tag antibody (Santa CruzBiotech, Cat. no. SC-803) diluted {fraction (1/500)} in 1% w/v BSA inPBS and agitated at room temperature for 30 minutes. After washing twicewith 0.0002% w/v Tween20 in PBS, each well was incubated with 100 μl ofdonkey anti-rabbit HRP (Jackson, Cat. no. 711-035-152) diluted {fraction(1/5000)} in 10% w/v BSA in PBS and agitated at room temperature for 30minutes. The plate was then washed 4 times with 0.0002% w/v Tween20 inPBS and developed as previously (Humphreys et al. 1996). ELISA to assessFab′ concentration was performed as described by Humphreys et al.(1996).

[0053] Accuracy of Cleavage of Signal Peptide in Front of AlkalinePhosphatase.

[0054] Periplasmic extracts were produced from E. coli expressing andsecreting alkaline phosphatase using the MCP3 signal peptide. Theseextracts were analysed by SDS-PAGE using 4-20% Trisglycine gels (Novex)according to manufacturers instructions. Proteins were transferred fromthe polyacrylamide gel to PVDF membrane (PSQ, Applied Biosystems), byelctroblotting in 10 mM CAPS (3-cyclohexylamino-1-propanesulfonic acid,Sigma) pH 11.0, then stained with Ponceau S. The band corresponding toalkaline phosphatase was excised, and the protein eluted for N-terminalsequence analysis.

[0055] b) Results.

[0056] Expression and Secretion of Alkaline Phosphatase TABLE 2 Alkalinephosphatase activity in liquid culture 3 hours post-induction. Alkalinephosphatase activity Signal peptide (ΔA₄₂₀ OD₆₀₀ ⁻¹ min⁻¹ ± S.D. n = 3)MCP1 0.91 ± 0.16 MCP2 4.90 ± 1.01 MCP3 4.82 ± 1.07 MCP4 4.68 ± 0.21 MCP54.31 ± 0.10 MCP6 4.27 ± 0.49 MCP7 4.21 ± 0.78 MCP8 3.61 ± 0.70 MCP9 3.52± 1.23 OmpA control 5.17 ± 0.58

[0057] Alkaline phosphatase expression was observed from all ten M13major coat protein signal peptide constructs, although some differencesin the level of expression were observed between constructs whose M13signal peptides were encoded by nucleic acid variants. In general thelevel of expression observed was similar to that obtained with thecontrol signal peptide OmpA (see Table 2 above).

[0058] A MCP3-containing clone expressing alkaline phosphatase wasarbitrarily chosen to assess the accuracy of signal peptide cleavage, asdescribed in Example 4 above. N-terminal sequencing revealed that thesignal peptide cleavage site had been correctly recognised resulting inthe correct N-terminal sequence for mature alkaline phosphatase.

[0059] Expression and Secretion of scFv

[0060] The ability of four of the variants as well as the native M13major coat protein signal peptide were assessed for their ability tosecrete scFv to the periplasm of E. coli by ELISA. The OmpA signalpeptide was also employed for comparative purposes. The results areshown in Table 3 below. TABLE 3 Yield of scFv in shake flask liquidculture 2.5 hours post-induction. scFv yield Signal peptide ng ml⁻¹OD₆₀₀ ⁻¹ ± S.D. (n = 3) MCP1 220.0 ± 63.7 MCP3 189.5 ± 17.1 MCP4 185.0 ±31.6 MCP8 185.5 ± 8.0  MCPn 264.0 ± 22.2 OmpA control 147.8 ± 14.0

[0061] Expression and Secretion of Fab″

[0062] The ability of the four variants assessed for their ability tosecrete scFv, as well as the native M13 major coat protein signalpeptide were also assessed for their ability to secrete Fab′ to theperiplasm of E. coli. Clones were grown and expression induced under thefermentation conditions described above. The yield of purifed Fab′ wasassessed by ELISA and the results are shown in Table 4 below. TABLE 4Yield of purified Fab' from fermentation. Yield of Purified Fab” Signalpeptide (mg/L) MCP1 35 MCP3 227 MCP4 383 MCP8 178 MCPn 94 OmpA control67

[0063] Again, the M13 Major coat protein was shown to be successful atexpressing and secreting high levels of Fab′. Surprisingly some of thenucleic acid variants were shown to be more efficacious than the OmpAcontrol. The results demonstrate that the level of expression can beincreased approximately five-fold over that produced by the controlsignal peptide by using in this instance the MCP4 variant nucleic acidto encode the M13 Major coat protein signal peptide.

[0064] Expression and Secretion of Different Fab′ Molecules

[0065] The M13 bacteriohage signal peptide can be used to direct thesecretion of different Fab′ molecules. To demonstrate this, the VH andVL regions in each of MCP1, MCP3, MCP4 and MCP8 Fab′ 40.4 constructswere excised as Eco RV-Spl I and Pvu II-Apa I fragments respectively,and replaced with similarly digested V_(H) and V_(L) regions from anantibody that recognises a different antigen to that recognised by Fab′40.4. Four master constructs were thus produced to enable the expressionof the new Fab′; each construct used a different nucleotide sequence toencode the M13 bacteriophage signal peptide in front of the light andheavy chains (NB the same nucleotide sequence was used for the light andheavy chain within a single construct) and each contained a light chainexpression cassette followed by a heavy chain expression cassette (seeFIG. 1C).

[0066] The expression and secretion of the Fab′ molecule (Fab′ 165) wasassessed under the fermentation conditions described in Example 4 above.The yield of purifed Fab′ was assessed by ELISA and the results areshown in Table 5 below: TABLE 5 Yield of different Fab' fragments fromfermentation Nucleotide sequence Yield of Purified encoding signalpeptide Fab' 165 (mg/L) MCP1 369 MCP3 134 MCP4 189 MCP8 437, 316

Example 5 Optimisation of Fab′ Expression

[0067] In the Examples described above, the nucleotide sequence encodingthe M13 bacteriophage signal peptide has been the same for both thelight and heavy chain in each Fab′ expression construct. In order toassess the effect, on expression and secretion, of combinations of thedifferent signal peptide encoding nucleotide sequences within the sameconstruct, two plasmid libraries were constructed. Each librarycontained all 16 possible combinations of the MCP1, MCP3, MCP4 and MCP8sequences in front of the light and heavy chains of Fab′ 165.

[0068] a) Construction of Fab′ Plasmid Library Containing VL ExpressionCassette Followed by VH Expression Cassette TABLE 6 The sixteen possiblecombinations of the different signal peptide coding sequence are shownin the matrix below. The first expression cassette is represented alongthe top, and the second expression cassette is represented down theside. These constructs all have a light chain expression cassettefollowed by a heavy chain expression cassette. HEAVY mcp1mcp1-VL-mcp1-VH mcp3-VL-mcp1-VH mcp4-VL-mcp1-VH mcp8-VL-mcp1-VH CHAINmcp3 mcp1-VL-mcp3-VH mcp3-VL-mcp3-VH mcp4-VL-mcp3-VH mcp8-VL-mcp3-VHmcp4 mcp1-VL-mcp4-VH mcp3-VL-mcp4-VH mcp4-VL-mcp4-VH mcp8-VL-mcp4-VHmcp8 mcp1-VL-mcp8-VH mcp3-VL-mcp8-VH mcp4-VL-mcp8-VH mcp8-VL-mcp8-VH

[0069] Starting with the four master Fab′ 165 constructs described inExample 4 above, these were digested with Pst I and Mfe I, and thefragments and vector backbones allowed to recombine randomly, thusresulting in all 16 possible combinations of signal peptide codingsequence as shown in Table 6.

[0070] b) Construction of Fab′ Plasmid Library Containing VH ExpressionCassette Followed by VL Expression Cassette.

[0071] Since Pst I is compatible with Nsi I and Mfe I is compatible withEco RI (see FIG. 2), each of the four Fab′ 165 master constructsproduced in Example 4 above, was treated as follows. A first sample ofeach construct was digested with Nsi I and Eco RI and the signal peptidecoding sequence and Heavy chain fragment was isolated. A second sampleof each construct was digested with Pst I and Mfe I, and both fragmentsisolated and purified. The Heavy chain fragment from the first digestionwas ligated with the vector backbone (lacking a light chain fragment)from the second, to produce a construct containing two heavy chains.This construct was then digested with Nsi I and Eco RI to remove thesecond signal peptide coding sequence and Heavy chain. The small Heavychain fragment was discarded and the remaining vector backbone(containing a 5′ Heavy chain fragment) was ligated to a mixture of thesignal peptide and light chain fragments from the second digestion.TABLE 7 The sixteen possible combinations of the different signalpeptide coding sequence are shown in the matrix below. The firstexpression cassette is represented along the top, and the secondexpression cassette is represented down the side. These constructs allhave a heavy chain expression cassette followed by a light chainexpression cassette. mcp1 mcp3 mcp4 mcp8 LIGHT mcp1 mcp1-VH-mcp1-VLmcp3-VHmcp1-VL mcp4-VH-mcp1-VL mcp8-VH-mcp1-VL CHAIN mcp3mcp1-VH-mcp3-VL mcp3-VH-mcp3-VL mcp4-VH-mcp3-VL mcp8-VH-mcp3-VL mcp4mcp1-VH-mcp4-VL mcp3-VH-mcp4-VL mcp4-VH-mcp4-VL mcp8-VH-mcp4-VL mcp8mcp1-VH-mcp8-VL mcp3-VHmcp8-VL mcp4-VH-mcp8-VL mcp8-VH-mcp8-VL

[0072] Thus a library of 16 constructs were produced having the order ofthe light and heavy chain expression cassettes reversed from that in thelibrary described in a) above. Table 7 shows the combinations of signalpeptides present in the library

[0073] c) Analysis of Fab′ 165 Expression

[0074] Expression studies were carried out in small scale shake flasksas described previously. Following on from the result obtained at thisscale, the ability of several of the different clones to express Fab′was assessed in fermentations, again as described previously. Levels ofexpression of light chains, heavy chains and total Fab′ was assessedusing surface plasmon resonance and/or ELISA.

[0075] Surface plasmon resonance binding assays were performed using aBIAcore™ 2000 instrument (Pharmacia Biosensor AB, Uppsala, Sweden).Murine IgG2a monoclonal anti-human IgG Pan Fd (CH1), obtained fromhybridoma HP6045 (ATCC) and murine IgG2a monoclonal anti-human kappalight chain constant domain (C_(κ)), obtained from hybridoma HP6053(ATCC) was immobilised onto CM5 sensor chips using standard NHS/EDCchemistry. Residual NHS esters were inactivated with ethanolaminehydrochloride (1M).

[0076] Fab′ fragments were captured by either an immobilised monoclonalanti-heavy chain or by an immobilised monoclonal anti-light chainantibody in separate flow cells. The presence of bound Fab′ was revealedby binding of the complementary monoclonal antibody (anti-light chain oranti-heavy chain) in a second step. High levels of immobilised antibodyensure that measurements are performed under mass transport-limitedconditions, where the contribution of the association rate constant tobinding is low in comparison to the contribution made by theconcentration of the Fab′ in the sample. The solution phase monoclonalantibody used in the second step is passed over the surface at a highconcentration so that binding is not limited by the association rateconstant of this interaction.

[0077] Assembled Fab′ fragments and correctly folded unassembled chainsare both detected during the first capture step. Binding of the secondantibody is only to an intact Fab′ fragment. Therefore, analysis of therelative binding at the first and second stages reveals the presence ofeither excess unassembled light chain, or excess unassembled heavy chainin the Fab′ sample and provides information on the stoichiometry ofassembly.

[0078] Assays were performed in both configurations for each sample, andeach sample was run in duplicate and in a randomised order.

[0079] (i) Where the concentration of assembled Fab′ was to bedetermined by light chain capture, samples and standards (10 μl at 10μl/min) were injected over immobilised HP6053, followed by a second stepin which HP6045 at 300 μg/ml was passed over the surface in the solutionphase.

[0080] (ii) Where the concentration of assembled Fab′ was to bedetermined by heavy chain capture, samples and standards (10 μl at 10μl/min) were injected over immobilised HP6045, followed by a second stepin which HP6053 at 500 μg/ml was passed over the surface in the solutionphase. In both cases, the surface was regenerated with 10 μl of 30 mMHCl at 30 μl/min. The number of resonance units determined using theBIAevaluation 3.1 (Pharmacia Biosensor AB), was read against a standardcurve. There was a linear response from 2 μg/ml down to 50 ng/mlpurified Fab′ standard.

[0081]FIG. 3 shows that the level of Fab′ expression varies considerablybetween different constructs having the expression cassettes in theheavy chain-light chain order. Similar results were obtained for lightchain-heavy chain library (data not shown). Thus the two librariescontaining different combinations of signal peptide coding sequences canbe used to optimise Fab′ expression. Using Eco RV-Spl I and Pvu II-Apa Idouble digestions, the light and heavy chains of other antibodies can besubstitute for those of Fab′ 165, and thus the libraries can be used tooptimise the expression of any Fab′ molecule.

[0082]FIG. 4 compares the yield of each chain and total Fab′ during thecourse of a fermentation run for various combinations of signal peptidecoding sequences. It is surprising that the yield of total Fab′ ismaximal when the levels of expression of heavy and light chains areclosely balanced. Thus Fab′ expression can be optimised using the signalpeptide libraries to achieve a balance between light and heavy chainexpression and this forms a further aspect of the invention,particularly when each signal sequence is under the control of its ownpromoter/operator.

[0083] REFERENCES:

[0084] Atlan, D. & Portarlier, R. 1984 Applied Microbiology &Biotechnology 19:5-12.

[0085] Fognini Lefebvre, N. & Portarlier, R. FEMS Microbiology Letters21:323 328.

[0086] Glover, D. M. 1995a. DNA cloning: a practical approach, VolumeII: Expression systems. IRL press.

[0087] Glover, D. M. 1995b. DNA cloning: a practical approach, VolumeIV: Mammalian systems. IRL press.

[0088] Humphreys, D. P., Weir, N., Mountain, A. & Lund, P. A. 1995.Journal of Biological Chemistry 270:28210-28215.

[0089] Humphreys, D. P. Weir, N., Lawson, A., Mountain, A. & Lund, P. A.1996 FEBS Letts. 380:194-197.

[0090] Humphreys, D. P., Vetterlein, O. M., Chapman, A. P., King, D. J.,Antoniw, P., Suitters, A. J., Reeks, D. G., Parton, T. A. H., King, L.M., Smith, B. J., Lang, V. & Stephens, P. E. (1998) Journal ofImmunological Methods 217:1-10.

[0091] Gray, G. L., Baldridge, J. S., McKeown, K. S., Heyneker, H. L. &Chang, C. N. 1985. Gene 39:247 254.

[0092] Kumagai, M. H., Shah, M., Terashima, M., Vrkljan, Z., Whitaker,J. R. & Rodriguez, R. L. 1990 Gene 94:209 216.

[0093] Sambrook, J. & Fritsch, E. 1989 Molecular cloning: a laboratorymanual. 2nd edition. Cold Spring Harbour Press, N.Y.

1. A method of producing an antibody chain or an antigen bindingfragment thereof, comprising culturing host cells containing anexpression cassette under conditions that result in expression of theantibody chain, or fragment thereof, from the expression cassette,wherein said expression cassette comprises a first nucleic acid encodinga bacteriophage signal peptide, or a variant thereof, operably linked toand in frame with a second nucleic acid encoding the antibody chain orantigen binding fragment thereof.
 2. A method according to claim 1,wherein the first nucleic acid encodes the bacteriophage M13 major coatprotein signal peptide, or a variant thereof.
 3. A method according toclaim 2, wherein the first nucleic acid encodes a variant of the M13major coat protein signal peptide with greater than 70% identity to thebacteriophage M13 major coat protein signal peptide.
 4. A methodaccording to claim 2, wherein the first nucleic acid encodes a variantof the M13 major coat protein signal peptide 75% identity to thebacteriophage M13 major coat protein signal peptide.
 5. A methodaccording to claim 2, wherein the first nucleic acid encodes a variantof the M13 major coat protein signal peptide with greater than 80%identity to the bacteriophage M13 major coat protein signal peptide. 6.A method according to claim 2, wherein the first nucleic acid encodes avariant of the M13 major coat protein signal peptide with greater than90% identity to the bacteriophage M13 major coat protein signal peptide.7. A method according to claim 2, wherein the first nucleic acid,encoding the native bacteriophage M13 major coat protein signal peptideamino acid sequence, differs in the nucleotide sequence from the nativeM13 nucleotide sequence.
 8. A method according to claim 7, wherein thefirst nucleic acid has the nucleotide sequence of MCP1, MCP3, MCP4,MCP5, MCP6, MCP7, MCP8, or MCP9.
 9. A method according to claim 7,wherein the first nucleic acid has the nucleotide sequence of MCP1,MCP3, MCP4, or MCP8.
 10. A method for producing a whole antibody or afragment thereof which comprises producing an antibody heavy and lightchain according to claims 1 to 9 and allowing the chains to assemble.11. A method according to claim 10 in which the heavy and light chainare produced in the same host cell from separate expression cassettes.12. A method according to claim 11 in which each expression cassette isunder the control of a single promoter/operator.
 13. A method accordingto claim 11 or claim 12 in which each signal peptide is selected toachieve a balanced expression of heavy and light chains.
 14. A methodaccording to any one of the preceding claims, which further comprises a)optionally, allowing the antibody, antibody fragment, antibody chain orantigen binding fragment to accumulate and b) isolating the antibody,antibody fragment, antibody chain or antigen binding fragment.
 15. Anucleic acid encoding the bacteriophage M13 major coat protein signalpeptide, or a variant thereof, for use in directing secretion of anantibody chain or antigen binding fragment thereof from the cytoplasm ofa prokaryotic host cell.
 16. A nucleic acid according to claim 15, whichencodes a signal peptide with greater than 70% identity to thebacteriophage M13 major coat protein signal peptide.
 17. A nucleic acidaccording to claim 15, which encodes a signal peptide with greater than75% identity to the bacteriophage M13 major coat protein signal peptide.18. A nucleic acid according to claim 15, which encodes a signal peptidewith greater than 80% identity to the bacteriophage M13 major coatprotein signal peptide.
 19. A nucleic acid according to claim 15, whichencodes a signal peptide with greater than 90% identity to thebacteriophage M13 major coat protein signal peptide.
 20. A nucleic acidencoding the bacteriophage M13 major coat protein signal peptide, whichdiffers in the nucleotide sequence from the wild-type nucleic acidsequence, but not in the amino acid sequence.
 21. A nucleic acidaccording to claim 20 wherein the nucleotide sequence is selected fromMCP1, MCP3, MCP4, MCP5, MCP6, MCP7, MCP8, or MCP9.
 22. A nucleic acidaccording to claim 21, wherein the nucleotide sequence of the M13 majorcoat protein signal peptide is selected from MCP1, MCP3, MPC4, or MCP8.23. An expression cassette comprising a first nucleic acid according toany one of claims 15 to 22, operably linked to and in frame with asecond nucleic acid encoding an antibody chain or antigen bindingfragment thereof.
 24. A vector comprising a nucleic acid according toany one of claims 15 to 22 or one or two expression cassettes accordingto claim
 23. 25. A host cell containing an expression cassette accordingto claim 23 or, a vector according to claim 24.